Purification of Coronavirus Proteins

The coronavirus proteins were produced and purified as previously described (15 (link), 48 (link), 49 (link), 50 (link)). Plasmids encoding N-terminal domain proteins were obtained from GenScript. Plasmids were transiently co-transfected in FreeStyle 293-F cells using 293Fectin (ThermoFisher). All cells were tested monthly for mycoplasma. DNA was prepared using a Midiprep kit (Qiagen). On day 5 or 6 post transfection cell culture supernatants were clarified by centrifugation and filtration with a 0.8-μm filter. Stepwise purification included affinity chromatography using StrepTrap HP (Cytiva) ran in 1X Buffer W (IBA Lifesciences) and eluting in 1X Buffer E (IBA Lifesciences), and by size-exclusion chromatography using Superdex 200 columns (Cytiva) in 10 mM Tris pH8, 500 mM NaCl.

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Pierre C.N., Adams L.E., Anasti K., Goodman D., Stanfield-Oakley S., Powers J.M., Li D., Rountree W., Wang Y., Edwards R.J., Munir Alam S., Ferrari G., Tomaras G.D., Haynes B.F., Baric R.S, & Saunders K.O. (2023). Non-neutralizing SARS-CoV-2 N-terminal domain antibodies protect mice against severe disease using Fc-mediated effector functions. bioRxiv.

Publication Preprint 2023

293Fectin Midiprep kit StrepTrap HP Superdex 200 columns

Affinity chromatography Buffer Cell culture Cells Centrifugation Coronavirus Filtration Mycoplasma Nacl Plasmids Proteins Size exclusion chromatography Transfection Tris

Corresponding Organization :

Other organizations : Duke University, University of North Carolina at Chapel Hill

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Variable analysis

independent variables

Plasmids encoding N-terminal domain proteins obtained from GenScript
Transient co-transfection of plasmids in FreeStyle 293-F cells using 293Fectin (ThermoFisher)

dependent variables

Expression and purification of coronavirus proteins

control variables

Cells were tested monthly for mycoplasma
DNA was prepared using a Midiprep kit (Qiagen)
Stepwise purification included affinity chromatography using StrepTrap HP (Cytiva) and size-exclusion chromatography using Superdex 200 columns (Cytiva)

controls

Positive controls not explicitly mentioned
Negative controls not explicitly mentioned

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